CRISPR/Cas9介导的同源重组技术构建猪肌抑素基因敲除细胞系

肌抑素是肌肉增生的负调控因子，为改良家畜产肉性状的重要候选基因。本研究设计了Cas9/sgRNA表达载体与供体DNA，通过电转染方法将其导入猪PK15细胞，经G418抗性筛选和荧光蛋白标记甄别，筛选到带阳性标记的细胞克隆。通过跨界PCR、长距离PCR、Western印迹、Southern印迹及PCR产物测序，证明了猪肌抑素的第3外显子序列特异性同源重组事件的发生。在猪肌抑素的第3外显子区域找到了1个有效的CRISPR/Cas9打靶位点，带阳性标记的细胞克隆经过多次筛选分离,获得了肌抑素单等位基因失活的稳定细胞系,为深入研究肌抑素的功能提供了重要的实验材料。

Generation of Porcine MSTN Knockout Cell Line Using CRISPR/Cas9-mediated Homologous Recombination

Myostatin(MSTN), as an inhibitor of muscle hyperplasia, is an important candidate gene which can improve meat traits in large domestic animals. Two kinds of designed vectors, MSTN Cas9 /sgRNA and donor DNA were delivered into porcine PK15 cell line by electric transfection. The Neo EGFP positive cell line was isolated in favor of the G418 resistance selection and Leica Fluorescent Microscope (Leica 4000B). Crossover PCR, long-distance PCR, Western blot, Southern blot and DNA sequencing were applied to detect the site specific homologous recombination event at the locus of exon3 of MSTN. In this study, an effective target site of CRISPR/Cas9 expression vector was found at the exon3 of MSTN, yielding a stable cell line harboring MSTN allelic mutant through multi screening and isolation. Our study provided the relevant experimental materials for the MSTN functional studies.